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PubMed1994

Characterization of the essential yeast gene encoding N-acetylglucosamine-phosphate mutase.

A previously cloned gene of Saccharomyces cerevisiae, which complements the growth defect of a phosphoglucomutase (pgm1 delta/pgm2 delta) double deletion mutant on a pure galactose medium [Boles, E., Liebetrau, W., Hofmann, M. & Zimmermann, F. K. (1994) Eur. J. Biochem. 220, 83-96], was identified as the structural gene encoding N-acetylglucosamine-phosphate mutase. The complete nucleotide sequence of the gene, AGM1, and surrounding regions were determined. AGM1 codes for a predicted 62-kDa protein with 557 amino acids and is located on chromosome V adjacent to the known gene PRB1 encoding protease B. No extended nucleotide or amino acid sequence similarities could be found in the databases, except for a small region of amino acids with high similarity to the active-site consensus sequence of hexosephosphate mutases. Three putative pheromone-responsive elements have been identified in the upstream region of the AGM1 gene. The gene is essential for cell viability. An agm1 deletion mutant progresses through only approximately five cell cycles to form a 'string' of undivided cells with an abnormal cell morphology resembling glucosamine auxotrophic mutants. Expression of the AGM1 gene on a multi-copy plasmid led to a significantly increased N-acetylglucosamine-phosphate mutase activity. Unlike over-expression of the AGM1 gene in a pgm1/pgm2 double deletion mutant which could restore phosphoglucomutase activity, over-expression of the PGM2 gene encoding the major isoenzyme of phosphoglucomutase did not increase N-acetylglucosamine-phosphate-mutase activity and did not restore growth of agm1 deletion mutant cells. Our observations indicate that the different hexosephosphate mutases of S. cerevisiae have partially overlapping substrate specificities but, nevertheless, distinct physiological functions.

Amino Acid Sequence↗

PubMed1997

The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase.

The function of UreC, the product of a 1,335-bp-long open reading frame upstream from the urease structural genes (ureAB) of Helicobacter pylori, was investigated. We present data showing that the ureC gene product is a phosphoglucosamine mutase. D. Mengin-Lecreulx and J. van Heijenoort (J. Biol. Chem. 271:32-39, 1996) observed that UreC is similar (43% identity) to the GlmM protein of Escherichia coli. Those authors showed that GlmM is a phosphoglucosamine mutase catalyzing interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, which is subsequently transformed into UDP-N-acetylglucosamine. The latter product is one of the main cytoplasmic precursors of cell wall peptidoglycan and outer membrane lipopolysaccharides. The present paper reports that, like its E. coli homolog glmM, the H. pylori ureC gene is essential for cell growth. It was known that growth of a lethal conditional glmM mutant of E. coli at a nonpermissive temperature can be restored in the presence of the ureC gene. We showed that complete complementation of the glmM mutant can be obtained with a plasmid overproducing UreC. The peptidoglycan content and the specific phosphoglucosamine mutase activity of such a complemented strain were measured; these results demonstrated that the ureC gene product functions as a phosphoglucosamine mutase. Homologs of the UreC and GlmM proteins were identified in Haemophilus influenzae, Mycobacterium leprae, Clostridium perfringens, Synechocystis sp. strain PCC6803, and Methanococcus jannaschii. Significant conservation of the amino acid sequence of these proteins in such diverse organisms suggests a very ancient common ancestor for the genes and defines a consensus motif for the phosphoglucosamine mutase active site. We propose renaming the H. pylori ureC gene the glmM gene.

Amino Acid Sequence↗

PubMed1983

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase.

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase, comprising 239 residues, was determined. The sequence was deduced from the four cyanogen bromide fragments, and from the peptides derived from these fragments after digestion with a number of proteolytic enzymes. Comparison of this sequence with that of the yeast glycolytic enzyme, phosphoglycerate mutase, shows that these enzymes are 47% identical. Most, but not all, of the residues implicated as being important for the activity of the glycolytic mutase are conserved in the erythrocyte diphosphoglycerate mutase.

Amino Acid Sequence↗

PubMed1975

Evidence for three enzymatic activities in one electrophoretic band of 3-phosphoglycerate mutase from red cells.

Electrophoresis of 3-phosphoglycerate mutase from erythrocytes of man and several animal species has been performed on cellulose acetate strips. In most cases the electrophoretic pattern of this enzymatic activity shows three bands. 2,3-diphosphoglycerate phosphatase and diphosphoglycerate mutase from erythrocytes of the same species have been revealed after migration during the same electrophoresis. We found that the band of 2,3-diphosphoglycerate phosphatase and the band of diphosphoglycerate mutase activities migrate at the same level as one of the bands corresponding to 3-phosphoglycerate mutase. Here, we discuss the possible existence of a single molecule carrying three enzymatic activities.

Animals↗

PubMed1998

The 2.3 A X-ray crystal structure of S. cerevisiae phosphoglycerate mutase.

The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined. This structure shows important differences from the lower resolution structure deposited in 1982. The crystal used to determine the new structure was of a different form, having spacegroup P2(1). The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction. The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts. Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides. The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase. However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal. An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge. The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure. Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.

Acid Phosphatase↗

PubMed2002

Structure and mechanism of action of a cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus with broad specificity phosphatase activity.

The crystal structure of Bacillus stearothermophilus PhoE (originally termed YhfR), a broad specificity monomeric phosphatase with a molecular mass of approximately 24 kDa, has been solved at 2.3 A resolution in order to investigate its structure and function. PhoE, already identified as a homolog of a cofactor-dependent phosphoglycerate mutase, shares with the latter an alpha/beta/alpha sandwich structure spanning, as a structural excursion, a smaller subdomain composed of two alpha-helices and one short beta-strand. The active site contains residues from both the alpha/beta/alpha sandwich and the sub-domain. With the exception of the hydrophilic catalytic machinery conserved throughout the cofactor-dependent phosphoglycerate mutase family, the active-site cleft is strikingly hydrophobic. Docking studies with two diverse, favored substrates show that 3-phosphoglycerate may bind to the catalytic core, while alpha-napthylphosphate binding also involves the hydrophobic portion of the active-site cleft. Combining a highly favorable phospho group binding site common to these substrate binding modes and data from related enzymes, a catalytic mechanism can be proposed that involves formation of a phosphohistidine intermediate on His10 and likely acid-base behavior of Glu83. Other structural factors contributing to the broad substrate specificity of PhoE can be identified. The dynamic independence of the subdomain may enable the active-site cleft to accommodate substrates of different sizes, although similar motions are present in simulations of cofactor-dependent phosphoglycerate mutases, perhaps favoring a more general functional role. A significant number of entries in protein sequence databases, particularly from unfinished microbial genomes, are more similar to PhoE than to cofactor-dependent phosphoglycerate mutases or to fructose-2,6-bisphosphatases. This PhoE structure will therefore serve as a valuable basis for inference of structural and functional characteristics of these proteins.

Amino Acid Sequence↗

PubMed1996

Isolation of a cDNA from tomato coding for an unregulated, cytosolic chorismate mutase.

A cDNA coding for chorismate mutase was isolated from tomato by complementing a chorismate mutase-deficient Escherichia coli strain with a cDNA library. Southern blot analysis suggests the existence of a single gene of this chorismate mutase type per haploid tomato genome. The abundance of the corresponding transcripts was highest in roots, lower in stems and cotyledons, and even lower in flowers and leaves. The activity of the protein expressed in E. coli was not regulated by the three aromatic amino acids. Characteristics of the sequence and of the enzymatic activity suggest that the identified cDNA encodes a cytosolic, unregulated CM-2 type chorismate mutase.

Amino Acid Sequence↗

PubMed1986

Wheat germ phosphoglycerate mutase: evidence for a metalloenzyme.

Wheat germ phosphoglycerate mutase, exposed to 3.4 M guanidinium chloride at 22 degrees C and pH 7.8, slowly undergoes time-dependent inactivation which can be fully reversed by adding excess Co2+ or Mn2+ to a 50-fold dilution of the denaturing medium. Titration of the denatured enzyme with either Co2+ or Mn2+ shows that wheat germ mutase preferentially binds Co2+. Assuming 1:1 complexation between metal atom and protein, the apparent dissociation constants (Kd) for E Co2+ and E Mn2+ at 22 degrees C and pH 8.7 are approximately 1.06 and 1.84, respectively. Other metal atoms (e.g., Cr2+, Cu2+, Fe2+, Fe3+, Mg2+, and Ni2+) have no effect in restoring the apoenzyme's catalytic activity. At low concentrations (0.11-0.23 mM) Zn2+ partially restores activity, but promotes protein precipitation at elevated concentrations. Evidence suggests that all bisphosphoglycerate-independent phosphoglycerate mutases require either an intra- or an extramolecular metal atom in order to function. Attempts to characterize wheat germ mutase as a glycoprotein have yielded negative results.

Bisphosphoglycerate Mutase↗

PubMed1987

Developmental expression of the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer and innervated by fetal rat spinal cord.

The electrophoretic pattern of phosphoglycerate mutase of adult innervated normal human muscle is composed predominantly of the muscle-specific isozyme, whereas the electrophoretic pattern of aneurally cultured human muscle is composed only of the brain-specific isozyme. We studied the transition of the isozymes (phosphogluterate mutase) in human muscle cultured in monolayer and innervated for 20 to 83 days by rat embryo spinal cord explants. In this culture system, regions of innervated muscle fibers in close proximity to the ventral part of the spinal cord explant continuously contracted and the contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated cultured human muscle fibers, the total activity of phosphoglycerate mutase was increased and the muscle-specific isozyme was expressed. The amount of muscle-specific isozyme directly correlated with the duration of innervation. This study demonstrated that expression of the gene for the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer is influenced by de novo innervation.

Animals↗

PubMed1994

Thyroid hormone stimulates phosphoglycerate mutase activity and isozyme transition in rat muscle tissues.

Triiodothyronine (T3) increases phosphoglycerate mutase (PGAM) specific activity in rat skeletal and cardiac muscles. This increase is concomitant with an increase in the proportion of phosphoglycerate mutase isozymes which contain type-M subunit. Propylthiouracil (PTU), an anti-hormone, not only decreases phosphoglycerate mutase activity with respect to control rats, but also decreases the total M subunit contents. In liver, which only possesses type-B subunit phosphoglycerate mutase, none of the effects were detected.

Animals↗

PubMed1990

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

Amino Acid Sequence↗

PubMed2004

Design, synthesis, and evaluation of aza inhibitors of chorismate mutase.

A series of aza inhibitors (4-9) of chorismate mutase (E.C. 5.4.99.5) was designed, prepared, and evaluated against the enzyme by monitoring the direct inhibition of the chorismate, 1, to prephenate, 2, conversion. None of these aza inhibitors displayed tighter binding to the enzyme than the native substrate chorismate or greater inhibitory action than the previously reported ether analogue, 3. Furthermore, no time-dependent loss of enzyme activity was observed in the presence of the two potentially reactive aza inhibitors (7 and 9). These results in conjunction with inhibition data from a broader series of chorismate mutase inhibitors allowed a novel proposal for the mechanistic role of chorismate mutase to be developed. This proposed mechanism was computationally verified and correlated with crystallographic studies of various chorismate mutases.

Aza Compounds↗

PubMed2003

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS.

Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range.

Bacillus subtilis↗

PubMed2006

1.6 A crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed.

The presence of exported chorismate mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric chorismate mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this chorismate mutase family, termed the AroQ(gamma) subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli chorismate mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.

Amino Acid Sequence↗

PubMed1997

Partial inactivation of chorismate mutase-prephenate dehydrogenase from Escherichia coli in the presence of analogues of chorismate.

Chorismate-5,6-epoxide, chorismate-5,6-diol, various adamantane derivatives and 2-hydroxy-phenyl acetate are structural analogues of chorismate that act as competitive inhibitors of both the chorismate mutase and prephenate dehydrogenase activities of the bifunctional enzyme, hydroxyphenylpyruvate synthase. The interactions of these chorismate analogues with both activities of the synthase are investigated further. Chorismate mutase and prephenate dehydrogenase activities were assayed spectrophotometrically at 290 and 340 nm, respectively. Data were fit by non-linear regression to appropriate equations describing the time-dependent formation of product or decay of enzymic activity. In the presence of these chorismate analogues, both the mutase and dehydrogenase activities undergo a time-dependent partial inactivation. Progress curves for synthesis of product by the mutase or dehydrogenase in the presence of chorismate-5,6-epoxide, chorismate-5,6-diol or adamantane-1,3-diacetate resemble time-courses characteristic of slow-binding inhibition. However, if the bifunctional enzyme was preincubated with a chorismate analogue prior to addition of substrate, only a minor proportion of enzymic activity was recovered, excluding the possibility of reversible, slow-binding inhibition. When hydroxyphenylpyruvate synthase binds certain chorismate analogues to form an EI complex, there is a slow conformational transition to an ET complex, which may be susceptible to oxidation leading to partial inactivation. Some protection against this inactivation is provided by high concentrations of dithiothreitol (20 mM), suggesting that the inactivation may be due to chemical oxidation.

Adamantane↗

PubMed1993

13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.

The chorismate mutase reaction is a rare enzyme-catalyzed 3,3-sigmatropic rearrangement of chorismate to prephenate. Bacillus subtilis chorismate mutase was overproduced and purified from Escherichia coli XL1-Blue (pBSCM2) using a modification of the procedure of Gray et al. (Gray, J. V., Grolinelli-Pimpaneau, B., & Knowles, J. R. (1990) Biochemistry 29, 376-383); the modification leads to minimal contaminating prephenate dehydratase activity (< 0.001%). The native molecular mass of B. subtilis chorismate mutase was determined by gel filtration to be approximately 44 kDa, indicative of a homotrimer of the 14.5-kDa subunits as determined by electrospray mass spectrometry. 13C NMR was used to study the structure of [U-13C]prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound 13C NMR resonances of [U-13C]prephenate were assigned, and where possible, 1JC,Cs were quantified; [1,3,5,8-13C]prephenate and [2,6,9-13C]prephenate, prepared respectively from [1,3,5,8-13C]chorismate and [2,6,9-13C]chorismate, aided the 13C NMR resonance assignments. Enzyme-bound prephenate exhibits remarkably different chemical shifts relative to free prephenate; the chemical shift changes range from -6.6 ppm for the C6 resonance to 5.6 ppm for the C5 resonance, suggesting a strong perturbation of the C5-C6 bond. 13C NMR studies of model compounds at various pH values and in various solvents suggest that the observed 13C chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pKas of the carboxylic acid groups or hydrophobic solvation at the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗

PubMed1992

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1991

pH dependency of the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli.

The variation with pH of the kinetic parameters associated with the mutase and dehydrogenase reactions catalyzed by chorismate mutase-prephenate dehydrogenase has been determined with the aim of elucidating the role that ionizing amino acid residues play in binding and catalysis. The pH dependency of log V for the dehydrogenase reaction shows that the enzyme possesses a single ionizing group with a pK value of 6.5 that must be unprotonated for catalysis. This same group is observed in the V/Kprephenate, as well as in the V/KNAD, profile. The V/Kprephenate profile exhibits a second ionizing residue with a pK value of 8.4 that must be protonated for the binding of prephenate to the enzyme. For the mutase reaction, the V/Kchorismate profile indicates the presence of three ionizing residues at the active site. Two of these residues, with similar pK values of about 7, must be protonated, while the third, with a pK value of 6.3, must be unprotonated. It can be concluded that all three groups are concerned with the binding of chorismate to the enzyme since the maximum velocity of the mutase reaction is essentially independent of pH. This conclusion is confirmed by the finding that the Ki profile for the competitive inhibitor, (3-endo,8-exo)-8-hydroxy-2-oxabicyclo[3.3]non-6-ene-3,5-dicarboxylic acid, shows the same three ionizing groups as observed in the V/Kchorismate profile. By contrast, the Ki profile for carboxyethyldihydrobenzoate shows only one residue, with a pK value of 7.3, that must be protonated for binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗